Toxicology

APES/Ms. Lolich

There are two parts to this activity: MSDS research and lab experiment BACKGROUND - PART A: LD50 AND MSDS
We handle many materials daily that are toxic. We are often unaware of the degree to which they are toxic. For a variety of reasons, different animals respond differently to the same toxin. Some animals may be very sensitive to a toxin, whereas others are relatively resistant to its effects. Because species of animals vary, it is important to understand that what is toxic to brine shrimp may not necessarily be toxic to other kinds of animals to the same extent. Many household items that we deal with on a regular basis are toxic materials, but we dont usually think of them as being toxic. It can be instructive to examine several such materials to determine their toxicity. The commonly used term to describe acute ingestion toxicity is LD 50. LD means Lethal Dose (deadly amount) and the subscript 50 means that the dose was acutely lethal to 50% of the animals to whom the chemical was administered under controlled laboratory conditions. The test animals (usually mice or rats) are given specific amounts of the chemical in either one oral dose or by a single injection and are then observed for 14 days. Since LD50 values are measured from zero up, the lower the LD 50 the more acutely toxic the chemical. Therefore, a chemical with an oral LD50 of 500 would be much less toxic than a chemical with an LD 50 of 5. LD50 values are expressed as milligrams per kilogram (mg/kg) which means mg of chemical per kg of body weight of the animal. Mg/kg is the same as ppm. For example, if the oral LD50 of the insecticide parathion is 4, a dose of 4 parts of parathion for every million parts of body weight would be lethal to at least half of the test animals. An MSDS (Material Safety Data Sheet) is a document (for each chemical) with information on all the physical and chemical properties for that chemical, as well as information on reactions and safe disposal of the chemical waste. The following information can usually be found in a MSDS: Identity of the organization responsible for creating the sheet and the date of issue. The material's identity, including its chemical and common names. Hazardous ingredients. Exposure limits. Physical and chemical hazards and characteristics. Health hazards. Emergency and first aid procedures. Spill and disposal procedures. Precautions and safety equipment.

1.

Data Table: Using your OWN MASS in kg, figure out how many total g would be required to kill 50% of perfect duplicates of yourself. Be careful about units! For your reference, a penny weighs around 3000 mg or 3 g. You don't need to show work for all of these problems, but write out ONE complete example of your conversion to LD50/person below the table so that I know how you did it. Remember, everyone's answers will be slightly different. Remember that 1lb. = .453 kg and 1kg = 2.2 lbs. Write these numbers on the line on the reverse side. 2. MSDS Research: Find a (MSDS) for an ingredient in some household substance you have (e.g. toothpaste, shampoo, mouthwash, etc.) MSDS's at the link from Ch. 19 webpage. Don't use any of the ones already listed below. Find the toxicity report section of the MSDS sheet. At the bottom of the back page, record the animal the test was done on, how test was administered (orally, inhalation, skin) and the # in proper units per/kg.

LD50 AND MSDS

Name __________________________ Student # _______ Points ________

My weight in lbs. = ___________ My weight in kg = ____________

Substance (source or product)

disodium EDTA (Secret) benzaldehyde (Cherry Flavor)* Tetrahydrocannabinol (THC from marijuana)* Ethyl acetate (Cherry Flavor)* propylene glycol (Cherry Flavor) Caffeine (Mountain Dew)* malic acid (sour candy)* Methanol (wood alcohol)* Nicotine (through mouth)* Botulinum toxin (bacteria)* potassium nitrate (fertilizer) sodium fluoride (toothpaste) parathion (pesticide Vx (nerve gas) tetrodotoxin (poison from puffer fish) diazinon (ant killer dust) amphetamine sulfate ephedrine gamma hydroxybutyrate (date rape drug)
Showing your work for ONE problem:

LD50 (mouse or rat) mg/kg or g/kg 2000. mg/kg 4.8 mg/kg 110 mg/kg 6100 mg/kg 20 g/kg 0.13 g/kg 1.6 g/kg 5628 mg/kg 190 mg/kg 3 x 10-8 mg/kg 190 mg/kg 52 mg/kg 6.0 mg/kg 2 x 10-2 mg/kg 334 x 10-6 g/kg 0.076 g/kg 32 mg/kg 0.600 g/kg 2.0 g/kg

LD50 for you mg/kg or g/kg

* natural substances My substance from home is ___________________ Ingredient researched is ____________________ Ld50 for a ___________________ administered ______________________is ___________________

BACKGROUND - PART B: BIOASSAY OF CHEMICALS ON BRINE SHRIMP

A bioassay is a toxicity test used to determine the dose or concentration of a toxicant. In dealing with toxins a frequent relative danger indicator is the LD-50. For example the LD-50 for sugar in rats is 30 grams, which is out of 100 laboratory rats, 50 would be expected to die at levels of 30 grams of sugar/kg of body weight. Nicotine has an LD-50 in rats of 0.05 g, which is much more toxic. A similar measure, the LC-50, (which stands for lethal concentration) is often used. In this lab we will use a small crustacean, the brine shrimp. It is normally found in brackish water and is a very hearty little organism - able to tolerate high salt concentrations.

Materials6 Test tubes 1 - Pipette (1 mL) Dissecting Microscope Net Rubber stopper Toxin (chosen from supply table) Brine (salt water in tank) Petri Dishes (6 per group) Test tube rack (in drawer 19) 10mL graduated cylinder (in blue cabinet) Brine Shrimp

Titration Method- (work in groups of 3 or 4, at the most)

1) Use a small amount of masking tape to label 5 test tubes as follows: 1:1, 1:10, 1:100, 1:1000, 1:10,000. 2) Take 11 mL of the full-strength material being tested for toxicity from the stock solution and add it to the test tube labeled 1:1. Place 9 mL of brine into each of the other test tubes. Brine is in the tank on the side counter. 3) Pipette 1 mL of "toxic" material from the 1:1 tube into the tube labeled 1:10. Mix well. 4) Pipette 1 mL from the 1:10 tube into the tube labeled 1:100. Mix well. 5) Pipette 1 mL from the 1:100 tube into the tube labeled 1:1000. Mix well 6) Pipette 1 mL from the 1:1000 tube into the tube labeled 1:10,000. Mix well. 7) Label 6 petri dishes as follows: 1:1, 1:10, 1:100, 1:1000, 1:10,000, control. Be sure to label the bottom of the dish, not the cover! Transfer all the test tube contents to the dishes. 8) Put 10 mL of brine in the control dish. Store the dishes in the box to continue the lab tomorrow. 9) You will check out with Ms. Lolich so the room is clean. Show her the bag of test tubes and stopper. Be sure to remove your tape from the tubes. Return graduated cylinder and test tube rack.

This is called serial dilution

Lab Day 1
1) Uncover all of your petri dishes. Use a CLEAN beaker, to dip into the brine shrimp tank. Using a pipette, move 10 brine shrimp into each Petri dish Petri dish and observe for 10 minutes. 2) Put any extra shrimp back into the tank for the other classes. 2) Use a dissecting microscope if needed, count the number of brine shrimp alive after 10 minutes. Record your data in Data Table A. 3) Recover your petri dishes and return your box to storage area until day 2. Wash and dry beaker to return to Ms. Lolich.

Lab Day 2
1) Uncover all of your petri dishes. 2) Use a dissecting microscope if needed, count the number of brine shrimp alive after 24 hours. Record your data in Data Table A. 3) Wash all the petri dishes out and dry them. Return the dishes stacked and covered to Ms. Lolich.

If you prefer concentration in percent versus part per (part per hundred, etc), the percents would be as follows: 1:1 = 100% (pollutant), 1:10 = 10%, 1:100 = 1%, 1:1000 = 0.1%, 1:10,000 = 0.01% Do not use both of the measurements!!!! Label your x-axis for which you choose. Data Table A Record observation in this table during your bioassay. Transfer them to your report for turn in. # brine shrimp alive after 10 after minutes 24 hours Graph Data Plot a line graph of dilution (X axis) vs. survival # (Y axis) using Excel. Determine the LD-50 from your graph. This graph should be included in your report. Computer savvy students can put it right into write up rather than attach as a separate sheet. Analysis 1. What is (are) the control(s) in this experiment? What is its purpose? 2. Based on your data in this lab, what is the safe concentration for brine shrimp Threshold Level of Toxicity (TTL)? 3. Often indicator species are used to study the overall health of an ecosystem. If you were to study an ecosystem containing brine shrimp, would you use it as an indicator species? Why or Why not? Explain your reasoning. 4. What possible sources of error were present in this experiment?

Lab Report Requirements

Each person in your group will hand in the following, stapled together and word-processed at the beginning of the hour on the due date. This means NOT after you arrive to class and print out your lab work: 1) A cover sheet with the name of the lab, your group members, your name and student #, the date of submission. Fill in the cover sheet the day you finish the lab!!!! 2) Standard lab write-up process from hypothesis through conclusion. 3) Data table (re-create Data Table A in Excel or copy from Ch. 19 on the website). 4) Linear graph of data using EXCEL. (or other graphing software). This cannot be hand done this time. If you need help, see Ms. Lolich for instructions. Link to create-a-graph is in Ch. 19 at my site. 5) Answers to the analysis questions as listed above. Be sure you have AP quality answers.